More than 8 million organic compounds are known and many are thought to be biodegradable by microorganisms, the principle agents for recycling organic matter on Earth. In this context, microbial enzymes represent the greatest diversity of novel catalysts. This is why microbial enzymes are predominant in industrial enzyme technology and in bioremediation, whether used as purified enzymes or in whole cell systems.
There is increased interest in engineering bacterial enzymes for improved industrial performance. For example, site directed mutagenesis of subtilisin has resulted in the development of enzyme variants with improved properties for use in detergents. Most applied enzymes, particularly those used in biodegrading pollutants, however, are naturally evolved. That is, they are unmodified from the form in which they were originally present in a soil bacterium.
For example, most bioremediation is directed against petroleum hydrocarbons, pollutants that are natural products and thus have provided selective pressure for bacterial enzyme evolution over millions of years. Synthetic compounds not resembling natural products are more likely to resist biodegradation and hence accumulate in the environment. This changes over a bacterial evolutionary time scale; compounds considered to be non-biodegradable several decades ago, for example PCBs and tetrachloroethylene, are now known to biodegrade. This is attributed to recent evolution and dispersal of the newly evolved gene(s) throughout microbial populations by mechanisms such as conjugative plasmids and transposable DNA elements.
A better understanding of the evolution of new biodegradative enzymes will reveal how nature cleanses the biosphere. Furthermore, the ability to emulate the process in the laboratory may shave years off the lag period between the introduction of a new molecular compound into the environment and the development of a dispersed microbial antidote that will remove it.
Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine)] is a widely used s-triazine (i.e., symmetric triazine) herbicide for the control of broad-leaf weeds. Approximately 800 million pounds were used in the United States between 1980 and 1990. As a result of this widespread use, for both selective and nonselective weed control, atrazine and other s-triazine-containing compounds have been detected in ground and surface water in several countries.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a recalcitrant compound that is transformed to CO2 very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27xc2x0 C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. High concentrations of atrazine, such as those occurring in spill sites have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is often detected in ground water and soils in concentrations exceeding the maximum contaminant level (MCL) of 3 xcexcg/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 parts per million (ppm)) atrazine have been found in the soil at spill sites more than ten years after the spill incident. Such point source spills and subsequent runoff can cause crop damage and ground water contamination.
There have been numerous reports on the isolation of s-triazine-degrading microorganisms (see, e.g., Behki et al., J. Agric. Food Chem., 34, 746-749 (1986); Behki et al., Appl. Environ. Microbiol., 59, 1955-1959 (1993); Cook, FEMS Microbiol. Rev., 46, 93-116 (1987); Cook et al., J. Agric. Food Chem., 29 1135-1143 (1981); Erickson et al., Critical Rev. Environ. Cont., 19, 1-13 (1989); Giardina et al., Agric. Biol. Chem., 44, 2067-2072 (1980); Jessee et al., Appl. Environ. Microbiol., 45, 97-102 (1983); Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995); Mandelbaum et al., Appl. Environ. Microbiol., 59, 1695-1701 (1993); Mandelbaum et al., Environ. Sci. Technol., 27, 1943-1946 (1993); Radosevich et al., Appl. Environ. Microbiol., 61, 297-302 (1995); and Yanze-Kontchou et al., Appl. Environ. Microbiol., 60, 4297-4302 (1994)). Many of the organisms described, however, failed to mineralize atrazine (see, e.g., Cook, FEMS Microbiol. Rev., 46, 93-116 (1987); and Cook et al., J. Agric. Food Chem., 29 1135-1143 (1981)). While earlier studies have reported atrazine degradation only by mixed microbial consortia, more recent reports have indicated that several isolated bacterial strains can degrade atrazine. In fact, research groups have identified atrazine-degrading bacteria classified in different genera from several different locations in the U.S. (e.g., Minnesota, Iowa, Louisiana, and Ohio) and Switzerland (Basel).
An atrazine-degrading bacterial culture, identified as Pseudomonas sp. strain ADP (Mandelbaum et al., Appl. Environ. Microbiol., 61, 1451-1457 (1995); Mandelbaum et al., Appl. Environ. Microbiol., 59, 1695-1701 (1993); de Souza et al., J. Bact., 178, 4894-4900 (1996); and Mandelbaum et al., Environ. Sci. Technol. 27, 1943-1946 (1993)), was isolated and was found to degrade atrazine at concentrations greater than about 1,000 xcexcg/ml under growth and non-growth conditions. See also, Radosevich et al., Appl. Environ. Microbiol., 61, 297-302 (1995) and Yanze-Kontchou et al., Appl. Environ. Microbiol., 60, 4297-4302 (1994). Pseudomonas sp. strain ADP (Atrazine Degrading Pseudomonas) uses atrazine as a sole source of nitrogen for growth. The organism completely mineralizes the s-triazine ring of atrazine under aerobic growth conditions. That is, this bacteria is capable of degrading the s-triazine ring and mineralizing organic intermediates to inorganic compounds and ions (e.g., CO2).
The genes that encode the enzymes for MELAMINE (2,4,6-triamino-s-triazine) metabolism have been isolated from a Pseudomonas sp. strain. The genes that encode atrazine degradation activity have been isolated from Rhodococcus sp. strains; however, the reaction results in the dealkylation of atrazine. In addition, the gene that encodes atrazine dechlorination has been isolated from a Pseudomonas sp. strain. See, for example, de Souza et al., Appl. Environ. Microbiol., 61, 3373 (1995). The protein expressed by the gene disclosed by de Souza et al., degrades atrazine, for example, at a Vmax of about 2.6 xcexcmol of hydroxyatrazine per min per mg protein. Although this is significant, it is desirable to obtain genes and the proteins they express that are able to dechlorinate triazine-containing compounds with chlorine moieties at an even higher rate and/or under a variety of conditions, such as, but not limited to, conditions of high temperature (e.g., at least about 45xc2x0 C. and preferably at least about 65xc2x0 C.), various pH conditions, and/or under conditions of high salt content (e.g., about 20-30 g/L), or under other conditions in which the wild type enzyme is not stable, efficient, or active. Similarly, it is desirable to obtain genes and proteins encoded by these genes that degrade triazine-containing compounds such as those triazine containing compounds available under the trade names; xe2x80x9cAMETRYNxe2x80x9d, xe2x80x9cPROMETRYNxe2x80x9d, xe2x80x9cCYANAZINExe2x80x9d, xe2x80x9cMELAMINExe2x80x9d, xe2x80x9cSIMAZINExe2x80x9d, as well as TERBUTHYLAZINE and desethyldesisopiopylatriazine. It is also desirable to identify proteins expressed in organisms that degrade triazine-containing compounds in the presence of other nitrogen sources such as ammonia and nitrate.
The present invention provides isolated and purified DNA molecules that encode atrazine degrading enzymes similar to, but having different catalytic activities from a wild type (i.e., from an isolated but naturally occurring atrazine chlorohydrolase). The term xe2x80x9caltered enzymatic activitiesxe2x80x9d is used to refer to homologs of atrazine chlorohydrolase having altered catalytic rates as quantitated by kcat and Km, improved ability to degrade atrazine, altered substrate ranges, altered activities as compared to the native sequence in aqueous solutions, altered stability in solvents, altered active temperature ranges or altered reaction conditions such as salt concentration, pH, improved activity in a soil environment, and the like, as compared with the wild-type atrazine chlorohydrolase (AtzA) protein.
In one preferred embodiment, the present invention provides DNA fragments encoding a homolog of atrazine chlorhydrolase and comprising the sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NOS:7-11 and SEQ ID NOS:17-21. In one embodiment the invention relates to these DNA fragments in a vector, preferably an expression vector. Further, the invention relates to the DNA fragment in a cell. In one embodiment the cell is a bacterium and in a preferred embodiment, the bacterium is E. coli. 
The invention also relates to s-triazine-degrading proteins having at least one amino acid different from the protein of SEQ ID NO:2, wherein the coding region of the nucleic acid encoding the s-triazine degrading protein has at least 95% homology to SEQ ID NO:1 and wherein the s-triazine-degrading protein has an altered catalytic activity as compared with the protein having the sequence of SEQ ID NO:2. In one embodiment, the protein is selected from the group consisting of SEQ ID NOS: 5, 6 and 22-26. In one embodiment the substrate for the s-triazine degrading protein is ATRAZINE. In another embodiment the substrate for the s-triazine degrading protein is TERBUTHYLAZINE and in yet another embodiment the substrate for the s-triazine degrading protein is MELAMINE. In another embodiment this invention relates to a remediation composition comprising a cell producing at least one s-triazine-degrading protein having at least one amino acid different from the protein of SEQ ID NO:2, wherein the coding region of the nucleic acid encoding the s-triazine degrading protein has at least 95% homology to SEQ ID NO:1 and wherein the s-triazine-degrading protein has an altered catalytic activity as compared with the protein having the sequence of SEQ ID NO:2. In a preferred embodiment the composition is suitable for treating soil or water. In another embodiment the remediation composition comprises at least one s-triazine-degrading protein having at least one amino acid different from the protein of SEQ ID NO:2, wherein the coding region of the nucleic acid encoding the s-triazine degrading protein has at least 95% homology to SEQ ID NO:1 and wherein the s-triazine-degrading protein has an altered catalytic activity as compared with the protein having the sequence of SEQ ID NO:2. In a preferred embodiment this composition is also suitable for treating soil or water. In one embodiment the remediation composition comprises the protein bound to an immobilization support. In yet another embodiment, these proteins are homotetramers, such as the homotetramers formed by AtzA.
In another embodiment the invention relates to a protein selected from the group consisting of proteins comprising the amino acid sequences of SEQ ID NOS: 5, 6 and 22-26.
In another aspect of this invention, the invention relates to a DNA fragment having a portion of its nucleic acid sequence having at least 95% homology to a nucleic acid sequence consisting of position 236 and ending at position 1655 of SEQ ID NO:1, wherein the DNA fragment is capable of hybridizing under stringent conditions to SEQ ID NO:1 and wherein there is at least one amino acid change in the protein encoded by the DNA fragment as compared with SEQ ID NO:2 and wherein the protein encoded by the DNA fragment is capable of dechlorination at least one s-triazine-containing compound and has a catalytic activity different from the enzymatic activity of the protein of SEQ ID NO:2. In one embodiment the s-triazine-containing compound is ATRAZINE, TERBUTHYLAZINE, or MELAMINE.
The invention also relates to a method for treating a sample comprising an s-triazine containing compound comprising the step of adding a protein to a sample comprising an s-triazine-containing compound wherein the protein is encoded by a gene having at least a portion of the nucleic acid sequence of the gene having at least 95% homology to the sequence beginning at position 236 and ending at position 1655 of SEQ ID NO:1, wherein the gene is capable of hybridizing under stringent conditions to SEQ ID NO:1, wherein there is at least one amino acid change in the protein encoded by the DNA fragment as compared with SEQ ID NO:2 and wherein the protein has an altered catalytic activity as compared to the protein having the amino acid sequence of SEQ ID NO:2. In one embodiment, the composition comprises bacteria expressing the protein. In one embodiment the s-triazine-containing compound is atrazine, in another the s-triazine-containing compound is TERBUTHYLAZINE and in another the s-triazine containing compound is (2,4,6-triamino-s-triazine). In one embodiment, the protein encoded by the gene is selected from the group consisting of SEQ ID NOS: 5, 6 and 22-26.
In another aspect, this invention relates to a method for obtaining homologs of an atrazine chlorohydrolase comprising the steps of obtaining a nucleic acid sequence encoding atrazine chlorohydrolase, mutagenizing the nucleic acid to obtain a modified nucleic acid sequence that encodes for a protein having an amino acid sequence with at least one amino acid change relative to the amino acid sequence of the atrazine chlorohydrolase, screening the proteins encoded by the modified nucleic acid sequence; and selecting proteins with altered catalytic activity as compared to the catalytic activity of the atrazine chlorohydrolase. Preferably, the atrazine chlorohydrolase nucleic acid sequence is SEQ ID NO:1. In one embodiment the altered catalytic activity is an improved ability to degrade ATRAZINE. In another embodiment, the altered catalytic activity is an altered substrate activity.
Other homologs with an improved rate of catalytic activity for atrazine include clones A40, A42, A44, A46 and A60 having nucleic acid sequences (SEQ ID NOS:17-21, respectively). Other homologs capable of better degrading TERBUTHYLAZINE include A42, A44, A46 and A60 as well as A11 and A13.